The method of obtaining2-globulin cystic fluid associated with the true disease

 

(57) Abstract:

The invention relates to medicine, in particular dermatology, namely the method of obtaining2globulin cystic fluid associated with the true disease. The essence of the invention is that a method of obtaining2globulin cystic fluid associated with the true disease, including deposition of proteins cystic fluid by ammonium sulfate, followed by dissolving it in 0.05 M phosphate buffer pH 7.4, then the product purified by the method of ion exchange chromatography on DEAE-cellulose, with selected fraction, eluroomus in a gradient from 0.2 to 0.6 M NaCl, method of hydrophobic chromatography on phenyl-sepharose 4B, selecting a faction in the gradient of 0.6 0 M (NH4)2SO4after dialysis subjected to gel filtration on Sephadex G-200, collecting the fraction with mol. m 50 70 KD and affinity chromatography on Con A-agarose, and the target product is a fraction, eluroomus in the concentration range of-D-glucopyranoside from 2.5 to 3.0% is the mole. m 60 10 KD, relative electrophoretic mobility of 0.83 + 0.02 and isoelectric point pI of 5.25 + 0,23. On the basis of a preparation obtained by the claimed method, it is assumed p is nuclear biological chemical (NBC fluid, associated with the true disease, supposed to be used as diagnosticum true bladderworts, and for monitoring and evaluating the quality of therapy. table 2.

The invention relates to medicine, namely to dermatology, namely the method of obtaining 2-globulin cystic fluid associated with the true disease.

The increase in the incidence of gallbladder dermatoses in the world, the obscurity of the cancellation mechanism of tolerance to membrane antigens spinous epithelial cells, the occurrence of acantholysis, epidermo-dermal separation of pathological processes formed the basis for the beginning of a series of new research studies on labeling of pathological processes when the true disease.

In this work the analysis of the spectrum of proteins cystic fluid of patients with hand, foot and study of physico-chemical properties of glikoproteid associated with the disease.

There are various ways to obtain proteins with electrophoretic mobility2-globulin from biological materials such as atherosclerotic changed the aortic wall. The method of obtaining this2-glikoproteid premonitory and methanol (V1:V2=3:1).

The residue is freeze-dried

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ion-exchange chromatography on DEAE-52 cellulose

Elution 0.5 M PA-phosphate buffer pH 7.0 in the range of NaCl concentration from 0.12 to 0.2 M

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gelfiltration on Sephadex G-150

Elution of protein fraction with M 8010 KD

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Affinity chromatography on lentil-lectin 413-sepharose

Elution with 2.5% solution of methylcellulose

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Drug2-glikoproteid aortic wall 91% purity

This method can be considered as only one of numerous analogues of the claimed method. The prototype in the available literature is not detected, because glycoprotein with electrophoretic mobility2-globulin from the cystic fluid obtained for the first time, although there is some experience of investigation of the protein spectrum of the cystic fluid.

The method is as follows. In this work, we used the cystic fluid obtained from several patients true disease.

The cystic fluid in the amount of 25 ml is treated with ammonium sulfate, taken at 50% saturation, the obtained precipitate was separated by centrifugation at 20000g, dissolved in 5 ml of 0.05 M phosphate buffer at pH 7.4, add 5 ml ohlazhdeniya. Concentrated preparation of purified according to the following scheme:

ion-exchange chromatography (DEAE 52 cellulose Watman ngland)

Select the faction eluroomus in the gradient NCl from 0.2 to 0.6 M

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Hydrophobic chromatography (henyl sepharose 4B harmacia-L)

Select the faction eluroomus in a gradient of ammonium sulfate from 0.6 to 0 (after this drug cialiswhat and concentrate to 1 ml)

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Gel filtration (Sephadex G-200, harmacia-L)

Select the mol fraction.m. 50-70 KD

< / BR>
Affinity chromatography (SOP-agarose Sigma AVE.)

Select the faction eluroomus in the concentration range of D-glycopyranoside from 2.5 to 3.0%

In this case, ion-exchange chromatography is performed on a column with DEAE-cellulose (Watman, England), balanced 0.05 M PA-phosphate buffer pH of 7.6 (working buffer) when the velocity of the flow 40 ml/h, hydrophobic chromatography is carried out in a column filled phenyl-separate 4B (L, Sweden), and balanced work buffer containing 1 M ammonium sulfate, when the velocity of the flow 40 ml/h, the gel-filtration is carried out in a column filled with Sephadex G-200 (harmacia, Sweden), balanced work buffer when the velocity of the flow 5 ml/h (for the determination of molecular weight gel filtration methodology is(harmacia, Sweden), equilibrated PA-acetate buffer pH 7.0 containing gl, l, nCl concentration of 1 mm, when the velocity of the flow 1 ml/h

Check out protein fractions during chromatography carried out using a flow-through photometer "Uvicord S" (L, Sweden), with subsequent immunochemical verification.

The amino acid composition was performed after hydrolysis of the drug 6 N. Hcl for 12, 24 and 36 h at 105 C in the amino acid analyzer "Durrum" DS-500 (AVE.).

The obtained purified product was used for immunization of rabbits of the chinchilla breed, in order to obtain monospecific antisera.

In the analysis of monospecific antisera method discountrates with subsequent immunoprevention shown that anticavity reveals cystic fluid one antigen with electrophoretic mobility2-globulin. Physicochemical properties and amino acid composition of the obtained protein is shown in table.1 and 2.

As can be seen from the table.1 the investigated protein has a relative mol.m. 743,6 KD, isoelectric point 4, 6, and electrophoretic mobility2-globulins.

In the structure of the protein part is dominated by Proline, glutamine is having in the molecule carbohydrate component.

Cleared2-globulin together with monospecific anticorodal to him was titrated with the goal of constructing a standard test systems.

Immunochemical analysis 2-globulin in different biological fluids of humans have shown that it is relatively specific for the cystic fluid obtained from patients true bladderwort, is not detected in the serum and spenomegaly fluid donors.

On the basis of a preparation obtained by the proposed method, it is possible to develop a method of obtaining a high-quality drug antibodies to it. Antibodies to2-globulin cystic fluid associated with the true disease, supposed to be used as diagnosticum true bladderworts, and for monitoring and evaluating the quality of therapy.

THE METHOD OF OBTAINING2-GLOBULIN CYSTIC FLUID ASSOCIATED WITH the TRUE DISEASE, including deposition of proteins cystic fluid by ammonium sulfate, followed by dissolving it in 0.05 M phosphate buffer pH 7,4, then the product purified by the method of ion exchange chromatography on DEAE-cellulose, with selected fraction, eluroomus in a gradient from 0.2 DV>2SO4after dialysis subjected to gel filtration on Sephadex G 200, collecting the fraction with mol.m. 50-70 KD and affinity chromatography on Con. -a agarose, and the target product is a fraction, eluroomus in the concentration range of - D glucopyranoside from 2.5 to 3.0% is the mole.m. 60 10 KD, relative electrophoretic mobility 0,83 0,02 and isoelectric point pJ of 5.25 0,23.

 

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